In Vitro Antibacterial and Antioxidant Activities, Pharmacokinetics, and In Silico Molecular Docking Study of Phytochemicals from the Roots of Ziziphus spina-christi

Ziziphus spina-christi (Rhamnaceae family) is a medicinal plant traditionally used to treat dandruff, wounds, hair loss, diarrhea, mastitis, abdominal pain, and gastrointestinal complications. To support this, the present work aims to study the in vitro antibacterial and antioxidant activities of compound isolates from the roots of Ziziphus spina-christi along with their in silico computational analyses. Compounds were isolated on silica gel column chromatography and an agar disc diffusion and DPPH radical scavenging assays were employed to study the antibacterial and antioxidant activities, respectively. The ADME and toxicity properties of the compounds were evaluated using SwissADME and ProTox-II online Web tools, respectively. Conversely, the in silico molecular docking studies were attained via a Biovia Discovery Studio Visualizer 2021 in combination with the AutoDock Vina software. The silica gel chromatographic separation of the combined CH2Cl2 : CH3OH (1 : 1) and CH3OH root extracts afforded trimethyl trilinolein (1), stearic acid (2), 13-hydroxyoctadeca-9, 11-dienoic acid (3), β-sitosteryl-3β-glucopyranoside-6′-O-palmitate (4), and stigmasterol (5). Notably, the in vitro antibacterial study revealed the extract and β-sitosteryl-3β-glucopyranoside-6′-O-palmitate (4) with the highest inhibitory activities (15.25 ± 0.35 and 14.25 ± 0.35 mm, respectively) against E. coli compared to ciprofloxacin (21.00 ± 0.35 mm) at 2 mg/mL. The CH2Cl2 : CH3OH (1 : 1) extract (IC50 : 1.51 µg/mL) and β-sitosteryl-3β-glucopyranoside-6′-O-palmitate (4) (IC50 : 5.41 µg/mL) also exhibited auspicious DPPH scavenging activities, followed by stigmasterol (5) (IC50 : 6.88 µg/mL) compared to the ascorbic acid standard (IC50 : 0.46 µg/mL). The molecular docking analyses unveiled the highest binding affinity by β-sitosteryl-3β-glucopyranoside-6′-O-palmitate (4) (−8.0 kcal/mol) against P. aeruginosa PqsA relative to the ciprofloxacin standard (−8.2 kcal/mol). Furthermore, the organ toxicity predictions showed that all the compounds exhibit no hepatotoxicity and cytotoxicity effects and stigmasterol (5) affords drug-likeness protocols. Overall, the combined experimental and computational investigations of this study support the traditional uses of Ziziphus spina-christi for antibacterial and natural antioxidant applications.


Introduction
Nowadays, considerable higher plants are cultivated globally to achieve valuable substances in the felds of medicine and pharmacy.Te pharmacological properties of plants gave rise to natural product-derived drugs made from some plants with medicinal advantages.Until the 18 th century, the medicinal activities of many plants, their consequence on human health, and their method of action were known.However, the active compounds were unknown [1].In Ethiopia, medicinal plants have been used in folk prescription against diferent ailments and their applications in traditional medicine have become an integral part of culture.Te traditional health practices and therapies are noted in oral tradition early religious manuscripts and traditional pharmacopoeias [2].Recent literature sources noticed that about 90% of livestock and 80% of the human population in Ethiopia rely on traditional prescriptions and traditional healers known by diferent local names are the primary players in the therapeutic aspects of traditional remedy practices [2].
Te genus Ziziphus (Rhamnaceae family) is comprised of about 100 species of deciduous trees and shrubs which are cultivated in the tropical and semitropical areas of the world [3].Among the traditionally useful species of the genus is Ziziphus spina-christi (Figure 1).Te plant is native to many parts of Africa, including Sudan, Ethiopia, Somalia, Eritrea, Chad, Kenya, Djibouti, Mali, Libya, Mauritania, Senegal, Nigeria, Tunisia, Algeria, and Zimbabwe, and exotic to Saudi Arabia, India, Israel, Comoros, Iran, Egypt, Iraq, Madagascar, Jordan, Morocco, Netherlands, United Arab Emirates, Syrian Arab Republic, and Zanzibar [4].Te traditional applications of Z. spina-christi have been studied worldwide for the treatment of numerous ailments [5].Traditional remedies from the roots, leaves, and fowers of the plant were reported to treat stomach pain in Sudan, Malawi, and Iran [6].In Bahrain, the extracts of the plant are used against wounds, dandruf, and hair loss [7].In Turkey and Palestine, the leaves and fruit's fber contents are used to treat skin infections, and constipation [8,9].In Sudan, the fruits are used as remedies for malaria, rheumatism, diarrhea, antispasmodics, and scorpion stings.Decoction is prepared by boiling the fruits and leaves in water for 30 min and then drunk as an oral complement to lower cancer and cholesterol risks [10].
In Ethiopia, Z. spina-christi is locally called "Qurqura" (Afan Oromo) and "Gaba" (Amharic and Tigrinya) and possesses substantial traditional uses.In Tigray (North Ethiopia), traditional healers use pounded leaves against dandruf, wounds, and hair loss.In some parts of the region, extracts of the leaves are also used to treat abdominal pain, diarrhea, mastitis, and gastrointestinal complications [11].Traditional healers around Kunama (North Ethiopia) also use the roots extract mixed with the roots or barks of Acacia oerfota, pound together, and mix the fne powder with water and then drink on an empty stomach to treat toothache, stomachache, and tumors [12,13].In Oromiya, the fruits have culinary purposes and are used as a remedy against throat infections and hepatitis [14].Te biological activity profles of Z. spina-christi were studied and literature surveys stated that the plant exhibits antiinfammatory [15], antioxidant [16], antibacterial [17,18], antimalarial [19], antifungal [20], and anticancer [21,22] activities with well-documented preparation techniques and mode of actions.Due to its content of tannins, it has signifcant antibacterial properties.Tis is because the tannins are linked with the proteins contributing to the metabolic inhibition inside the microorganism and helps to remove it [23].Treatment of eye infections, headaches, bone pain, colon cramps, scorpion stings, rheumatism, and strengthening the immune system are also other health benefts of the plant [24,25].

Biochemistry Research International
Te recent progress in computational methods has critically introduced the rationale for designing and identifying therapeutically active natural products that can target proteins of interest.Te natural molecules isolated from plants can be evaluated by in silico approaches to verify their medicinal possibilities [34].Te in silico study is a computerbased innovative approach of analysis by enabling research activities without the need of experimental works.In this context, computational analyses have become indispensable in analyzing natural compounds and developing novel drugs.Tis is because in silico study infuences the entire drug discovery trajectories with cost and time bargains [35].In silico studies are mainly performed along with in vitro investigations, both to verify the potential activities of natural products.In toxicity study, this ofers an overview of the use of in silico approaches in testing the cytotoxicity of compound isolates and confrms the knowledge about chemical structures and their medicinal implications [36,37].Tus, this study was supported by in silico analyses to validate the antibacterial and antioxidant properties of isolated natural products from the roots of Z. spina-christi.
To the best of our knowledge, there are limited previous phytochemical and biological activity studies on the roots of the plant despite the fact that it is used by Ethiopian traditional healers to cure various infectious diseases.So, inspired by the ethnobotanical profle, phytochemistry, and pharmacological evidence of the plant, this study aims to investigate the in vitro antibacterial and antioxidant values of the phytochemicals from the roots extract of Z. spina-christi along with their computational studies.

Plant Material.
Roots of Z. spina-christi were collected from the Adama Science and Technology University campus and its surrounding areas, Adama, Ethiopia, in October 2022.Te plant was identifed by a taxonomist (Mr. Melaku Wendafrash), and its specimen (HZS007) was deposited at the Herbarium of Addis Ababa University, Ethiopia.Te sample materials were washed with distilled water and airdried for one month at room temperature without direct exposure to sunlight.Finally, the plant material was pounded into fne powder using an electric blender (Shanghai Jinkle, Scientifc Instrument, China) and stored in a polyethylene bag until extraction.

Instrumentation.
Extraction of the plant material (maceration) was performed on an orbital shaker (VRN-200, Taiwan) and the extracts were concentrated in a rotary evaporator (DW-RE-3000, China).Te root extracts and fractions were chromatographed on silica gel using column chromatography (column size; 500-725 mm) and eluted with increasing solvent polarity.TLC was used for purity analysis and spots were visualized on a UV lamp (UV4AC6/ 2, China), sprayed with 1% vanillin-sulphuric acid reagent followed by direct heating at 100 °C.Melting points of the compounds were determined on a Japson-type apparatus (JA90161, India).Te IR spectral data were generated with the help of a Perkin Elmer Bx infrared spectrometer equipped with KBr pellets and a UV-Vis spectrophotometer (CE4001, Cambridge, UK) was used for antioxidant activity assay.NMR spectral results were performed using 600 MHz Bruker AVANCE III NMR instruments and the residual signals of the solvent (7.28 ppm (δ H ) and 77.2 ppm (δ C ) for CDCl 3 ) were used as a reference.For antibacterial activity tests, a hood equipped with UV-radiation and laminar airfow, a micropipette (1000 µL), an incubator, an autoclave, and Petri dishes (90 mm) were utilized.( Part of the crude extract (22.5 g) was chromatographed on silica gel column chromatography (180 g, 60-200 mesh as stationary phase) and eluted with increasing gradient of ethyl acetate in n-hexane followed by methanol in chloroform ratio, successively.A total of 175 fractions (25 mL each) were collected and their purity was monitored with the help of TLC and UV lamp.Te retention factor (R f ) values of each pure fraction were calculated and fractions with similar R f values were mixed.Finally, the structural information of the isolated compounds was established following the data obtained from the IR and NMR instruments.Fractions 25-27 obtained with 20% ethyl acetate in n-hexane were combined and purifed using the gradient elution of nhexane up to 40% ethyl acetate in n-hexane to aford trimethyl linolein (1) (28.4 mg).In addition, fractions 70-80 isolated with 30% ethyl acetate in n-hexane were subjected to Biochemistry Research International gradient elution with 20% ethyl acetate in n-hexane up to 60% ethyl acetate in n-hexane to yield stearic acid (2) (36.0 mg).13-hydroxyoctadeca-9, 11-dienoic acid (3, 22.0 mg) was isolated from fractions 81-94 using the isocratic elution of 40% ethyl acetate in n-hexane mixture.Fractions 105-124, collected using 60% ethyl acetate in nhexane were combined and purifed with the gradient elution of 20% ethyl acetate in n-hexane up to 80% ethyl acetate in n-hexane to obtain β-sitosteryl-3β-glucopyranoside-6′-O-palmitate (4) (26.4 mg).Furthermore, fractions 50-62 isolated using 30% ethyl acetate in n-hexane were mixed and purifed with the isocratic elution of 30% ethyl acetate in n-hexane as eluents to aford stigmasterol (5) (36.2 mg) (Scheme 1).[38][39][40] using a commercial ciprofoxacin antibiotic as a positive control.A stock solution of the CH 2 Cl 2 : MeOH (1 : 1) extract (50 mg/ mL) and each compound (2 mg/mL) were prepared in 4% DMSO followed by the preparation of 25 and 12.5 mg/mL (crude extract) and 1 and 0.5 mg/mL (isolated compounds).Each solution (100 µL) was loaded onto the sterilized paper discs using a micropipette and transferred to the Petri dishes comprising the cultured bacterial strains.For complete difusion, the Petri dishes were left for half an hour and incubated at 37 °C for 24 hrs.Te measured diameters of inhibition zones (mm) reveal the antibacterial efciency of the test samples.

In Vitro
2.6.In Vitro Antioxidant Activity Tests.Te antioxidant efcacies were performed using in vitro 2, 2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging assay as per the previous protocol [41] using ascorbic acid and sample-free DPPH as positive and negative controls, respectively.Stock solutions (1000 μg/mL) of the compound isolates, extract, and ascorbic acid standard were prepared in CH 3 OH and were further diluted into four diferent concentrations (500, 250, 125, and 62.5 μg/mL) using a dilution method.To each prepared concentration, a fresh DPPH solution (1 mL, 0.04% w/v in CH 3 OH) was added.Te sample solutions were incubated for 30 min and their absorbances were determined using the UV-Vis spectrophotometer in triplicate at 517 nm.Te scavenging potentials were calculated using equation ( 2) and the results were reported as mean ± SEM.Finally, the IC 50 values were calculated from the relationship curves.

DPPH radical scavenging activity
where A and A ∘ revealed absorbances of the sample and DPPH radical in the prescribed solvent, respectively.

In Silico Drug Likeness and Pharmacokinetic Analyses.
Te drug-likeness predictions were established by adopting Lipiniski's rule of fve [42], and the top pharmacokinetic characteristics of the compound isolates were computed using the SwissADME (https://www.swissadme.ch/)online Web tool [43].Furthermore, the ProTox-II online Web Server (https://tox-new.charite.de/)was applied to establish the organ toxicity properties of the compounds [44].

Molecular Docking Studies.
In order to have an understanding of the right binding pocket of the ligands with the protein targets and analyze the observed in vitro experimental results [45], two of the compound isolates with promising in vitro activities were subjected to molecular docking studies and compared with reference drugs.Molecular docking parameters, such as hydrogen bonds, binding afnity (kcal/mol), and residual amino acid interactions along with 2D and 3D structural depictions of the most stable conformations were provided.Accordingly, the compounds were docked against four bacterial protein targets namely, S. aureus PK (PDB ID: 3T07), S. pyogenes 10782 streptopain (PDB ID: 6UKD), DNA gyrase B of E. coli (PDB ID: 6F86), and PqsA of P. aeruginosa (PDB ID: 5OE3) and a single human enzyme namely, human myeloperoxidase (PDB ID: 1DNU).Te bacterial protein targets were chosen based on their metabolic signifcance to the bacteria and the similarity of the compound isolates to the cocrystallized ligands in the protein complexes.

Protein Preparation.
Te protein preparations were performed following the reported standard protocol [46].Initially, the crystal structures of the protein targets were retrieved from the Protein Data Bank (https://www.rcsb.org) using their PDB identifcations.Te 3D crystal structures were imported into Biovia Discovery Studio Visualizer 2021 followed by the removal of water molecules, heteroatoms, and complexes bound to the receptor molecules.To compute a site-specifc molecular docking, ligand groups were selected from the co-crystallized protein structures and defned, and the binding sites were retrieved by identifying the x, y, and z coordinates from the SBD site spheres.Te active sites were recognized by studying the binding interaction of the ligand and the receptor and the dockings were computed on the active sites of the prepared receptors.Tereby, the dimensions of the SBD site sphere of each target protein were established as follows: PDB ID: 3T07 (0.014, 0. .989)Å for x, y and z coordinates, respectively.Subsequently, polar hydrogens were added for the correct ionization of the amino acid residues [47,48].Finally, the energy of the proteins was minimized using the CHARMM Force Field steepest descent algorithm with a maximum number of 1000 steps at a root mean square (RMS) gradient of 0.01.Te energy minimization process was continued until the protein fulflled a convergence gradient of 0.001 kcal•mol −1 [46].Biochemistry Research International

Ligand Preparation.
Te structures of the compound isolates were drawn on the ChemOfce tool (Chem Draw 16.0) and assigned with suitable 2D orientations.By applying the ChemBio3D tool, energy minimizations were performed with the molecular modeling (MM2) algorithm until it fulflled the RMS gradient of 0.01 kcal•mol −1 to achieve the minimum energy conformers.Lastly, the ligand molecules with minimized energy were saved in PDB format and used as an input for the AutoDock Vina to perform molecular docking simulations [46].

Protein-Ligand Docking Strategy.
Te docking protocol was validated to confrm the reliability and accuracy of the molecular docking outcomes, as per previous protocols [46,49,50].Te objective was to precisely reproduce the docking interactions and binding pores of the co-crystallized ligands inside the experimentally crystallized structure of proteins.Tus, the original ligand of the co-crystallized protein was separated and prepared for the molecular docking of the ligands of interest.Te ligands of interest were then docked back into the active sites of the protein using the AutoDock Vina 4.2 software, and all the saved fles were run via Command Prompt to generate the afnity and root mean square deviation (RMSD) values [47].Notably, the RMSD values ranging from (0-2) Å were suitable for the molecular docking demonstrating that the protocol is feasible for other compound inhibitors [50].For each ligand scored, nine diferent poses were generated and the conformation of the most stable binding afnity and RMSD (small binding scores) was preferred to study the interactions between the ligands and receptor.Finally, the image preparations, ligand interactions, and orientations were computed using the Biovia Discovery Studio Visualizer 2021 [51][52][53].
2.9.Spectral and Statistical Data Analysis.Te IR and NMR data were manipulated using Origin 8.0 and MestReNova softwares, respectively.Te in vitro antioxidant and antibacterial data were tabulated in a Microsoft Excel 2013 spreadsheet, and the values were reported as percent of scavenging activity and mean ± standard error of the mean (SEM), respectively.Te antibacterial activities were deduced by the relative comparison of the inhibition zones of the compounds and extract with the standard antibiotic (ciprofoxacin).
Notably, the 13  (from DEPT-135 spectrum) are evident that one of the olefnic moiety of the fatty acid group exhibits an additional methyl substituent (Supplementary Materials, Table S2, and Figure S3).In addition, the types of carbons were verifed by the DEPT-135 spectrum which revealed three olefnic signals assignable to nine carbons, one oxymethine, and two oxymethylene signals, twelve sp 3 methylene signals assignable to thirty-six carbons, and two methyl signals assignable to six carbons in accordance with the 13 C NMR spectrum of the compound (Supplementary Materials, Table S2, and Figure S4).Finally, the spectral data agreed with the literature values [54,55] of trimethyl trilinolein (1) (Figure 2).
Compound 2 (36.00 mg, R f : 0.63 in 20% ethyl acetate in n-hexane, mp: 69-71 °C) was obtained as white crystals.It was UV inactive and a vanillin-sulphuric acid spray was applied on the TLC plate followed by heating on a hot plate at 110 °C for visualization.In its FTIR spectrum, a broad absorption band observed between 3500 and 2554 cm −1 suggested the presence of a carboxylic acid functional group.In addition, the strong absorption bands at 2918 and 2842 cm −1 correspond to the -C-H stretching frequencies in the aliphatic region.Te spectrum also showed absorption bands at 1705 and 1167 cm −1 attributed to -C�O and -C-O stretching frequency of carboxylic acids, respectively (Supplementary Materials, Table S3, and Figure S5).
Te 1 H NMR (600 MHz, CDCl 3 ) spectrum of the compound displayed triplet signals at δ 2.37 (2H, t, J � 7.6) and 0.90 (3H, t, J � 7.0) attributed to the methylene protons at C-2 and terminal methyl protons (C-18) of a fatty acid skeleton, respectively.Te spectrum also exhibited multiplet peaks at δ 1.66 (2H, m) and 1.28-1.32(26H, m) assignable to C-3, and C-4 up to C-17 of the fatty acid chain, respectively (Supplementary Materials, Table S4, and Figure S6).In its 13   S4, and Figure S7).Notably, the DEPT-135 spectrum revealed one methyl signal at δ 14.1 and sixteen methylene signals which were consistent with the 13 C NMR spectral analysis of the compound (Supplementary Materials, Table S4, and Figure S8).Overall, the data generated match with previous work of Abdurrahman and Cai-Xiab [56] for stearic acid (2) (Figure 2).Te compound was also identifed from the GC-MS analysis of the volatile oils of Z. spina-christi and its structural information was supported by mass fragmentation patterns (Supplementary Materials, Figure S9).
Te 13 C NMR (150 MHz, CDCl 3 ) spectrum revealed forty-fve well-resolved carbon signals assignable to ffty-one carbons attributed to a steroidal skeleton with additional glucose and fatty acid moieties.Te fatty acid moiety was confrmed by the presence of an ester carbonyl at δ 174.7 (C-1″) and the type of the fatty acid chain was verifed as palmitic acid based on the acid-catalyzed methanolysis followed by GC-MS analysis.Te signals at δ 140.3 and 122.2 assignable to C-5 and C-6, respectively, indicated the presence of two olefnic carbons of which the frst δ value corresponds to an sp 2 quaternary carbon (also supported by DEPT-135 spectrum).Te spectrum also showed the resonance of six sugar signals at δ 101.2 (C-1′), 76.1 (C-3′), 73.9 (C-5′), 73.6 (C-2′), 70.1 (C-4′), and 63.4 (C-6′) of which the signal of the anomeric carbon resonates at δ 101.2.Another oxymethine signal observed at δ 79.7 attributed to the carbon atom forming a glycoside linkage (C-3) in the main skeleton of the structure.In addition, the carbon signals at δ 56.8, 56.1, 50.2, 45.8, and 36.2 correspond to the sp 3 methine carbons of the structure and were assignable to C-14, C-17, C-9, C-24, and C-20, respectively.Te spectrum also showed two sp 3 quaternary carbons at δ 42.4 (C-13) and 36.8 (C-10) supported by the DEPT-135 spectrum.Te values of the remaining methylene and methyl signals are clearly stated in the supporting information (Supplementary Materials, Table S6, and Figure S14).In agreement with the 13 C NMR spectral data, its DEPT-135 spectrum displayed one sp 2 methine, six oxymethines, one oxymethylene, seven sp 3 methines, nineteen sp 3 methylenes assignable to twenty-fve carbons, and seven methyl signals (Supplementary Materials, Table S6 and Figure S15).Moreover, the COSY spectrum showed correlations for two spin systems.Accordingly, 3 J correlations were observed between H-2″ (δ 2.31) and H-3″ (δ 1.61) (Supplementary Materials, Table S6, and Figure S16).Finally, the data generated match with the reported values [58] of β-sitosteryl-3β-glucopyranoside-6′-O-palmitate (4) (Figure 2).

Antibacterial Activity.
Te agar disc difusion evaluation results revealed that the crude extract and isolated compounds exhibited auspicious antibacterial activities against the bacterial strains.Te choice of bacterial pathogens was based on their prevalence and availability in Ethiopia.Te average zones of inhibition (mm) against the growth of the bacterial pathogens are presented in Table 1 and the activities showed smooth relationships with concentration.Accordingly, the crude extract and isolated compounds displayed better activities at 50 and 2 mg/mL, respectively.For the CH 2 Cl 2 : CH 3 OH (1 : 1) extract, the highest inhibition zone (15.25 ± 0.35 mm) was observed at 50 mg/mL against E. coli compared to ciprofoxacin (21.00 ± 0.35 mm) and the lowest activity (9.50 ± 0.70 mm) was displayed at 12.5 mg/mL against P. aeruginosa.Our results align with previous reports against diferent bacterial strains.Previous work on methanol extracts of the roots and leaves of Z. spina-christi revealed promising activities against E. coli (15.00 ± 0.50 mm) and P. aeruginosa (12.00 ± 1.00 mm), respectively [61].In a related study by Ads et al. [32], the chloroform extract from the stem bark of Z. spina-christi displayed substantial antibacterial efcacy against E. coli (15.90 ± 0.63 mm), S. aureus (16.40 ± 1.20 mm), and P. aeruginosa (15.30 ± 1.50 mm) and the activities match with the results of the present work.

In Silico Pharmacokinetic Predictions.
In the present study, Lipinski's rule of fve [42] and Veber's rule [63] were applied to predict the drug-likeness properties of the isolated Biochemistry Research International compounds.

Compounds
with MW < 500 Daltons, NHA < 10, NHD < 5, and LogP (iLogP) <5 satisfy Lipinski's rule of 5 and are good drug candidates.In addition, predicted compounds with TPSA < 140 and NRB < 10 satisfy Veber's rule and aford good oral bioavailability [47,48].Generally, therapeutic agents with high molecular weight   Biochemistry Research International (>500 Daltons) decrease absorption by reducing their concentrations in the intestinal epithelial surface leading to passive difusion across the bilayer membrane [64,65].Tus, drug candidates with MW < 500 Daltons are suggested in accordance with Lipiniski's rule of fve.Tree of the investigated compounds in this study exhibited molecular weight within the recommended range of drug-like compounds as presented in Table 3. Compounds with zero or one violation are chosen for drug candidacy, thereby 13hydroxyoctadeca-9, 11-dienoic acid (3) satisfed Lipinski's rule with no violation.Fewer violations (1) were also predicted for stearic acid (2), and stigmasterol (5) which aford the concept of drug candidacy.Only stigmasterol (5) obeyed Veber's rule with NRB � 5 and TPSA � 20.23 Å2 and hence safe absorption by the gut.Te LogP value provides information on the afnity of compounds to lipophilic properties [66].Tus, stearic acid (2) and 13-hydroxyoctadeca-9, 11-dienoic acid (3) exhibited <5 values of LogP suggesting their optimum lipophilicity (Table 3).Te ADME properties of the isolated compounds along with ciprofoxacin are also portrayed in Table 3. Te LogKp (cm/s) value of 13-hydroxyoctadeca-9, 11-dienoic acid (3) (−4.19) was predicted better than the others relative to ciprofoxacin (−9.09).Te smaller the LogKp (cm/s), the lower the skin permeability of the compound [43].Trimethyl trilinolein (1) revealed a noninhibitor of all the cytochromes and was in accordance with the prediction results of ciprofoxacin (Table 3).

Conclusion
In the present study, fve compounds were isolated from the combined CH 2 Cl 2 : CH 3 OH (1 : 1) and CH 3 OH root extracts of Z. spina-christi, and their in vitro antioxidant and antibacterial activities along with in silico computations were performed.Te crude extract and compound isolates exhibited substantial activities against the bacterial strains.In addition, the DPPH radical scavenging activities revealed auspicious scavenging potential for CH 2 Cl 2 : CH 3 OH (1 : 1) extract (IC 50 : 1.51 µg/mL) and β-sitosteryl-3β-glucopyranoside-6′-O-palmitate (4) (IC 50 : 5.41 µg/mL) compared to ascorbic acid (IC 50 : 0.46 µg/mL).Te organ toxicity predictions of the compounds revealed that stearic acid (2) and 13-hydroxyoctadeca-9, 11-dienoic acid (3) were inactive towards all types of toxicities.Te molecular docking fndings in combination with the in vitro experimental data ofer prospects for the advancement of novel therapeutic agents to combat various human ailments.Tus, the in vitro antioxidant and antibacterial studies together with the computational analyses suggest the potential use of the extract and stigmasterol (5) as antibacterial and antioxidant agents, and we recommend further investigations on other biological activities of Z. spina-christi and the compound isolates to consolidate its traditional welfares.

2. 4 .
Extraction and Isolation.Te pounded sample of Z. spina-christi (450 g) was extracted by maceration technique in 2.25 L (1 g : 5 mL) of CH 2 Cl 2 : CH 3 OH (1 : 1) followed with an equal volume of CH 3 OH for 3 days each with continuous shaking at room temperature.A Whatman No. 1 type flter paper was employed for fltration of the extracts over gravity fltration, and the fltrates were dried in a rotary evaporator at 40 °C under a reduced pressure.Lastly, the extraction yields (%) were calculated using the following equation: Extraction yield(%) � Weight of the crude extract Weight of the powdered sample * 100.